The benefit of flow cytometry is the rapid simultaneous measurement of several parameters on a cell by cell basis. Uncompensated polychromatic analysis of mitochondrial. Flow cytometry within reach the analytical power and versatility of todays laserbased flow cytometry systems have. Jc10 mitochondrial membrane potential assay kit flow. Stepbystep detailed flow cytometry protocol enquire bio. Changes in m, reflected by aggregation level of jc1, can be determined as a ratio of red to green mean fluorescence intensities using flow cytometry. Here, we present a protocol that uses jc1 dye to assess the mmp of cells after being exposed to hr. Double fluorescence staining of mitochondria by jc1, either as. Note the different fluorescence intensity of the two cell types, due to the presence of a higher number of mitochondria in monocytes. Flow cytometry is a means of identifying and measuring certain physical and chemical characteristics of cells or particles as they travel in suspension.
Cell preparation for flow cytometry thermo fisher scientific. Jc1 mitochondrial membrane potential assay gbiosciences. Discover more with our introduction to flow cytometry. Flow cytometry within reach the analytical power and versatility of todays. Flow cytometric analysis of isolated liver mitochondria to. L of 4,6diamidino2phenylindole dapi to exclude dead cells by flow cytometry. Store the jc 1 reagent at 20c, protected from light and moisture preferably in a desiccator. Adjust the cell suspension to a concentration of 1 x 10 7 cellsml with cold 4 o c. General considerations and theory of flow cytometry 2.
Improving the accuracy of flow cytometric assessment of. Culture cells to the desired density generally not. Even at 1 m concentration, jc1 tends to precipitate in aqueous buffer. Flow cytometric analysis of mitochondrial membrane potential.
Flow cytometry is the technical process that allows for the individual measurements of cell fluorescence and light scattering. Determination of high mitochondrial membrane potential in. Flow cytometry fcm is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles in this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. Jc1 could also be used in fluorescent flowcytometry studies of the mitochondrial. Preparation of cells for flow cytometry for further information. Measurement of jc 1 fluorescence in s, r and t cells by fluorescence cytometry. Looking at jc1 staining it appears that flow cytometry.
We first examined whether the fcca with the mitochondrial membrane depolarization sensor, 5, 50, 6, 60tetrachloro 1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide jc 1. Cell meter jc10 mitochondrion membrane potential assay. Mitoprobe jc1 assay kit for flow cytometry thermo fisher. Flow cytometric analysis of mitochondrial membrane. Membrane potential was measured using rhodamine123 rh123 717, 3,3 dihexyloxacarbocyanine iodide dioc 6 3 18,19, or 5,5,6,6 tetrachloro1,1,3,3 tetra. Jc1 mitochondrial membrane potential detection kit abp. Hl60 cells were seeded and labeled according to section 11. Has anyone used jc1 for flow cytometry analysis of isolated mitochondria. The following protocol describes the preparation steps for the use of the jc1 dye on cultured cells. Effects of the incubation with the depolarizing agent valinomycin on d y. Fluorescence signal from pacific blue, alexa fluor 488, and alexa fluor 647 clickit edu flow cytometry assay kits. Analysis of the mitochondrial membrane potential using the. The reagents in this kit are sufficient for staining up to 500 samples for jc1. Jc1 compensation for flow cytometry how to prepare biological single stained.
Has anyone used jc1 for flow cytometry analysis of isolated. I am attempting to look at mitochondrial membrane potential in monocytes using the jc1 cationic dye. Applications of flow cytometry to clinical microbiology. Ac jc 1 fluorescence is seen in both the fl2 and fl 1. Cells were then treated for 4 hours with a titration series of the thiazolidinedione troglitazone and both monomer and aggregate forms were read on a perkin elmerwallac 1420 victor 2 multilabel plate reader. Changes in m reflected by different forms of jc1 as either green or red fluorescence can be determined as a ratio of green. Changes in m, reflected by aggregation level of jc 1, can be determined as a ratio of red to green mean fluorescence intensities using flow cytometry. Nov 22, 2012 investigation of fluorescence of jc 1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard.
Individual experimental designs for flow cytometry. Whole blood samples may be used undiluted unless the cell count is high, e. Flow cytometry of apoptosis unit 18 university of illinois. Besides its use in flow cytometry, jc10 can also be used in fluorescence imaging. Bd mitoscreen flow cytometry mitochondrial membrane.
Flow cytometry is now a widely used method for analyzing the. Tetraethylbenzimidazolylcarbocyanine iodide jc1 by using fluorescenceactivated flow cytometry. Quantitative assay by flow cytometry of the mitochondrial. Flow cytometry ploidy determination, cell cycle analysis, dna content per nucleus sergio j. This initial step helps to confirm that flow cytometry is the correct. Jc 1 mitochondrial membrane potential assay gbiosciences. Ac jc1 fluorescence is seen in both the fl2 and fl1 channels r1 in the.
Flow cytometry is now a widely used method for analyzing the expression of cell surface and intracellular. The loss of mitochondrial membrane potential is a hallmark for apoptosis. A flow cytometric assay to quantify invasion of red blood cells by rodent plasmodium parasites in vivo. Flow cytometry flow cytometry is the technical process that allows for the individual measurements of cell fluorescence and light scattering. Although most flow cytometry experiments involve labeling populations of cells that are relatively abundant, the number of cells required will vary depending upon the rarity of your cells. Comparison of jc1 and mitotracker probes for mitochondrial. Flow cytometric chemosensitivity assay using jc1, a sensor. Jc10 is developed to be a superior alternative to jc1 where high dye concentration is desired. Run bone marrow sample and acquire at least 1 x 10 6 events.
This unit presents very recent developments in both fluorescent probes and functional applications and demonstrates the use of the jc. Jc1 compensation for flow cytometry how to prepare biological. Adjust the cell suspension to a concentration of 1 x 10 7 cellsml with cold 4 o c pbsbsa buffer. Once reconstituted, full activity is guaranteed for 6. Flow cytometry protocols flow cytometry guide biorad. In some cell lines jc10 has even superior performance to jc1. Caymans jc1 mitochondrial membrane potential assay kit can be used to study mitochondrial behavior in a variety of conditions, including apoptosis. Investigation of fluorescence of jc1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at. An overview of the stepbystep protocol that illustrates the design of a highdimensional spectral flow cytometry panel. We have developed a protocol to use jc10 in a fluorescence microplate platform. Comparison of jc1 and mitotracker probes for mitochondrial viability assessment in stored canine platelet concentrates. Although jc1 is widely used in many labs, its poor water solubility causes extraordinary inconvenience. Mtr also presented strong correlation with the metabolic parameters analyzed.
Investigation of fluorescence of jc 1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on. Mp 34152 mitoprobe jc 1 assay kit for flow cytometry product information revised. I am attempting to look at mitochondrial membrane potential in monocytes using the jc 1 cationic dye. As the name implies, flow cytometry is the measurement of cells in a flow system, which delivers the cells singly past a point of measurement. Oct 25, 2007 three different gates were designed on the dot plot of jc 1 monomers versus jc 1 aggregates to identify cells with a different mmp first row, from the top and to separately analyze their.
This process is performed at rates of thousands of cells per second. Jc 1 staining of peripheral blood lymphocytes and monocytes. In modern flow cytometry, the way you start an experiment remains the same. Jc1 dye for mitochondrial membrane potential thermo. Detection of the mitochondrial membrane potential by the. Comparison of jc 1 and mitotracker probes for mitochondrial viability assessment in stored canine platelet concentrates. Cell meter jc10 mitochondrion membrane potential assay kit. Double fluorescence staining of mitochondria by jc 1, either as green fluorescent jmonomers or as red fluorescent jaggregates, was used for monitoring the mitochondrial membrane potential by flow cytometry figure 1 a. For intracellular staining, refer to the best protocols section of the ebioscience website for the staining intracellular antigens for flow cytometry protocols. Cells were stained with jc1 according to the protocol and analyzed on a bd facscalibur as described in the section methods for staining cells with jc1 and analyzing by flow cytometry. Compared to jc1, jc10 has much better water solubility. Jc1 mitochondrial membrane potential assay kit ab1850. Cell preparation for flow cytometry protocols invitrogen ebioscience reagents red blood cell lysis protocols using ebioscience lysis buffers invitrogen ebioscience reagents staining cell surface targets for flow cytometry invitrogen ebioscience reagents. Flow cytometry ploidy determination, cell cycle analysis.
Jc1 mitochondrial membrane potential flow cytometry. Design and optimization protocol for highdimensional. This process is performed at rates of thousands of cells per. In general, researchers will stain between 1 x 10 5 and 1. Methods for staining cells with jc1 and analyzing by flow. Flow cytometry intracellular staining protocol fix cells before intracellular staining to fixing and permeabilization ensure stability of soluble antigens or antigens with a short halflife see the special. Oct, 20 the purpose of the study is to establish a simple and relatively inexpensive flow cytometric chemosensitivity assay fcca for leukemia to distinguish leukemic blasts from normal leukocytes in clinical samples. Detected by jc 1 assay, the mmp of cells is independent of factors such as mitochondrial size, shape, and density that may influence singlecomponent fluorescence signals 14. This kit is suitable for the flow cytometric detection of mitochondrial membrane potential in mammalian cells and for screening apoptosis inhibitors and activators. Culture cells in appropriate 6, 12 or 24well cell culture plates a. General intracellular target flow cytometry protocol using. A flow cytometric assay to quantify invasion of red blood. Jc1 compensation for flow cytometry how to prepare biological single stained controls. Preparation of human peripheral blood mononuclear cells pbmc leukocytes are the most commonly analyzed cells in flow cytometry.
Consequently, the results of jc1 assay are relatively reliable. Jc 1 compensation for flow cytometry how to prepare biological single stained controls. Jc 1 dye can be used as an indicator of mitochondrial membrane potential in a variety of cell types, including myocytes and neurons, as well as in intact tissues and isolated mitochondria. Jc 1 mitochondrial membrane potential detection kit provides a quick and reliable method to detect the mitochondrial membrane potential changes in apoptotic cells using flow cytometry. Flow cytometry facs protocols psr the bd facscalibur platform allows users to perform both cell analysis and cell sorting in a single benchtop system. The higher the ratio of red to green fluorescence, the higher the polarization of the mitochondrial membrane. Filtration of the sample just before flow assays is recommended. Flow cytometry protocol for intracellular targets using detergents. We have adapted a flow cytometry method for the quantitative measurement of m which utilizes the lipophilic, cationic, fluorescent probe 3,3. The following protocol describes introducing jc1 reagent into the cultured cells and analyzing the stained cells by flow cytometry.
The example below is a protocol for labeling with annexin vallophycocyanin. Cells were stained with jc 1 according to the protocol and analyzed on a bd facscalibur as described in the section methods for staining cells with jc 1 and analyzing by flow cytometry. This unit presents very recent developments in both fluorescent probes and functional applications and demonstrates the use of the jc 1 probe for measuring mitochondrial membrane potential by flow cytometry. This book provides instructions and guidelines for performing modern flow cytometry that should render experiments that are more successful. Jc 10 mitochondrial membrane potential assay kit ab1123 is designed for use with flow cytometry, and it provides the most robust assay method for monitoring changes in mitochondrial membrane potential. Store the jc1 reagent at 20c, protected from light and moisture preferably in a desiccator. Im new to flow cytometry and any suggestion would be appreciated. Clickit edu protocol for flow cytometry thermo fisher. Avoid subjecting the reconstituted reagent to multiple freezethaw cycles by dispensing. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, jc. Detected by jc1 assay, the mmp of cells is independent of factors such as mitochondrial size, shape, and density that may influence singlecomponent fluorescence signals 14. For each dilution the parasitaemia was measured using the hoechstjc 1 flow cytometry method described above as well as by using hoechst 33342 or jc 1 fluorescence alone. Caymans jc 1 mitochondrial membrane potential flow cytometry assay kit can be used to study mitochondrial behavior in a variety of conditions, including apoptosis.
Here, we present a protocol that uses jc 1 dye to assess the mmp of cells after being exposed to hr. Flow cytometry applications national jewish health. Principle this kit utilizes jc10, a superior alternative to jc 1. Once reconstituted, full activity is guaranteed for 6 months. Cuttingedge and comprehensive, flow cytometry protocols, fourth edition is a valuable resource for researchers and scientists who are interested in continuing andor expanding their knowledge of flow cytometry. Both colors can be detected using the filters commonly mounted in all flow cytometers. The ratio of red to green fluorescence of jc1 is dependent only on membrane potential, and not influenced by mitochondrial size, shape, or density. Jc1 mitochondrial membrane potential flow cytometry assay kit. We developed a new protocol in which cells are equilibrated with very low dye concentrations 1 of 8 introduction to flow cytometry flow cytometry is a popular laserbased technology. Direct and indirect staining, staining of intracellular antigens, permeabilization and cell preparation protocols the flow cytometry protocols below provide detailed procedures for the treatment and staining of cells prior to using a flow cytometer. Mitochondrial membrane potential, jc1 dye, fluorescent probe, cationic, flow cytometry. Flow cytometric analysis of mitochondrial membrane potential using jc 1 article in current protocols in cytometry editorial board, j.
Using three sources of excitation and polychromatic flow cytometry, we have developed a protocol that can be applied to cells undergoing apoptosis. Besides its use in flow cytometry, it can also be used in fluorescence imaging and fluorescence microplate platform. This information can be used to individually sort or separate subpopulations of cells. Tetraethylbenzimidazolylcarbocyanine iodide jc 1 by using fluorescenceactivated flow cytometry. Set up the gating strategy to identify the different hematopoietic populations figure 2. A flow cytometrybased assay for measuring mitochondrial. Jc1 compensation for flow cytometry how to prepare. Principles and applications of flow cytometry table of. You must define the hypothesis or biological question that you want to answer. Changes in membrane potential have long been known to represent early activation events. Cell meter jc10 mitochondrion membrane potential assay kit optimized for flow cytometry assays, protocol. Although jc 1 is widely used in many labs, its poor water solubility causes extraordinary inconvenience. Bd mitoscreen flow cytometry mitochondrial membrane potential.
Jurkat human tcell leukemia cells were treated with 10. Ac jc 1 fluorescence is seen in both the fl2 and fl 1 channels r1 in the. M, jc 1 remains in the monomeric form, which exhibits green fluorescence. Caymans jc1 mitochondrial membrane potential flow cytometry assay kit can be used to study mitochondrial behavior in a variety of conditions, including apoptosis. Mitoprobe jc1 assay kit for flow cytometry 2 most widely implemented application of jc1 is for detection of mitochondrial depolarization occurring in apoptosis figure 2. Flow cytometry protocols thermo fisher scientific us. We offer invitrogen jc 1 dye as a bulk chemical and in the invitrogen mitoprobe jc 1 assay kit, optimized for flow cytometry. Flow cytometric analysis of isolated liver mitochondria has been described previously by several investigators. Note the relevant shift towards the lower right part of. The system supports a wide variety of research and clinical applications and is complemented by a broad suite of intuitive software solutions to.
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